DNA-protein biding often results in global changes in the DNA topology, such as bending or kinking. For DNA to bend, there needs to be adjustments in the structural units that define the duplex conformation. The overall DNA conformation is defined by many factors, one of which is the "pucker" preference of the ribose ring. While the furanose ring of a simple nucleotide is in dynamic equilibrium between a South (S) sugar pucker (2'-endo, B DNA-like) and a North (N) sugar pucker (3'-endo, A DNA/RNA-like), upon incorporation into a DNA strand, the furanose ring adopts a preferred conformation. In a typical B-like DNA duplex, the base pairs involved in a topological adjustment such as a bend assume an altered, more A-like (N) sugar pucker. Prearrangement of the DNA duplex to more closely resemble the bound state ("bent" conformation) may increase the binding affinity or decrease the disassociation energy from a protein of interest. As outlined in project Z01 BC 006174, the preparation of unique synthetic nucleotide analogues based on a bicyclo [3.1.0] hexane template system has been refined and the conformation of the monomers studied. This modified scaffold can lock the sugar pucker in either an N or S conformation depending on the relative position of the base on the [3.1.0] scaffold. Modified N- thymidine and N-adenine nucleotides were inserted into the Dickerson Drew dodecamer (5'-CGCGAATTCGCG-3'), a prototypical B-type DNA. In the last annual report, we had completed an NMR study using residual dipolar couplings (RDCs) on the DNA's incorporating N-locked thymidines and showed that these modifications caused bending of the duplex depending on the nature and number of modifications added. We also have preliminary evidence from single atom neutron scattering (SANS) experiments that the DNA does bend when N-locked thymidines are built into the strand and this work is continuing with other modified DNAs. We have repeated the biophysical studies on these oligomers, critically examined the corresponding N-locked adenine-substituted oligomers and examined six DNA's with either or both S-locked thymidines or adenine incorporation by circular dichroism (CD), Differential scaning calorimetry (DSC) and nuclear magnetic resonance (NMR). Melting temperatures for two of the N-locked adenine derivatives were higher that the native dodecamer suggesting a paradoxical stabilization by addition of A-like monomers. However, our assertion that preorganization with S-locked monomers (B-like DNA conformation) would more efficiently facilitate assembly of and stabilize the duplex was incorrect based on the complete set of biophysical data that we now have available for all six of the se modified DNA's. We found that there was a concentration-dependent formation of a second unstable species formed in most of these derivatives by DSC, NMR and CD measurements. Melt data by both ultraviolet (UV) and DSC measurements were complementary and confirmed this result. We have assigned the NMR spectra of these derivatives and are proceeding with the measurement of RDCs based on our newly developed method. In addition, we have assembled tables of the distance data for the N-locked thymidine analogues and these are being analyzed and compared with data obtained by extensive molecular modeling studies. The idea mentioned in the last report of remaking building blocks with specific 13C labeling for enhanced sensitivity in the NMR experiments has not been realized but is still part of the long range plan of the research.